PROJECT SUMMARY Despite increased survivorship among patients, breast cancer remains the second leading cause of cancer- related death in women. Associated mortality is most often due to metastatic disease. The magnitude of this problem provides a strong impetus for studies that may lead to the development of new chemopreventative strategies and/or lifestyle changes to prevent and treat cancer metastasis. In this regard, epidemiologic studies have found that elevated circulating cholesterol is a risk factor for breast cancer metastasis. On the other hand, patients taking inhibitors of HMG-CoA reductase (statins) demonstrate increased recurrence free survival. The observation that the circulating concentrations of a primary metabolite of cholesterol, 27-hydroxycholesterol (27HC), are correlated with those of cholesterol, together with the knowledge that this metabolite can serve as a ligand for both the estrogen receptors (ERs) and liver X receptors (LXRs), suggests a potential mechanistic link between hypercholesterolemia and breast cancer metastasis. Indeed, we have found that 27HC robustly increases metastasis in several murine models of mammary cancer, while metastasis is reduced in mice lacking the capacity to synthesize 27HC. Intriguingly, we have found that the pro-metastatic effects of 27HC are due to its ability to impact the host immune system. Furthermore, extracellular vesicles released from immune cells treated with 27HC possess pro-metastatic properties. However, the precise receptor-mediated mechanisms by which 27HC influences immune cells or extracellular vesicle cargo remain unknown. We hypothesize that 27HC alters the immune micro-environment via selective modulation of the ERs and LXRs culminating in the increased cancer cell colonization of distal sites. We will systematically test this hypothesis by (1) identifying the receptor-mediated mechanisms responsible for the actions of 27HC including the determination of selective modulation of the ERs and LXRs by different oxysterols, (2) evaluating the mechanisms by which 27HC regulates extracellular vesicle cargo and release, and (3) elucidating the mechanisms by which 27HC influences immune cells to facilitate metastasis. A series of integrated experiments will determine both the cellular and biochemical mechanisms behind the 27HC promotion of metastasis. This information will determine the best approaches for targeting the ER and LXR axes within immune cells, important information given the recently initiated clinical trials targeting these receptors. It is anticipated that this line of investigation will lead to the near-term development of new strategies to treat and prevent metastatic breast cancer. This research is of significant importance, given the high prevalence of hypercholesterolemia, stark differences in the effects of ligands for the ERs and LXRs, and that the majority (>90%) of mortality associated with breast cancer is due to metastatic disease.